Many new rapid assays for microorganisms have been developed during the last decade. For example, two of the most sensitive tests on the commercial market are based on nucleic acid probes and the Polymerase Chain Reaction (PCR) assay. Most other tests such as radioimmunoassay and ELISA require significantly higher levels of specific bacteria for enumeration. Thus, when samples with low counts are assayed by RIA or ELISA, an enrichment step is necessary.
Nucleic acid probes consist of DNA sequences from a target bacteria that are used to detect homologous sequences of bacterial DNA or RNA. Generally, the DNA of the probe contains a radioisotope (e.g. 32P, 3H, 125I, or 14C) or other reporter molecule. When the probe DNA hybridizes with the bacterial DNA/RNA, hybridization can be detected. Detection limits can be as low as 104 cfu/g, but are best when detecting 106 to 107 cfu/g. Nucleic acid probe based methods are problematic in the limited number of probes, the sample preparation time, enrichment time, and the complexity of the process. Thus this detection method has not been utilized by commercial industries or medical laboratories.
PCR techniques more suited to identification of microorganisms rather than enumeration, but detection limits may be as low as a single cell in purified media even in a mixed bacterial cell population. However, some compounds impede the amplification process of PCR techniques and limit the sensitivity to greater than 104 cfu/ml. For example, PCR hybridization has been reported to be impeded in the presence of fermented solids such a cheese or whey. Also, many PCR assays require a 12 hour or overnight pre-enrichment step plus the time for hybridization. The complexity of the hybridization step tends to limit the procedure to a sophisticated facility such as a research laboratory. Thus the process time and complexity of the process limits the use of this procedure by many industrial and medical laboratories.
Factors that affect the commercial use of a rapid assay include: difficulty of sample preparation, time required to enrich the bacteria in question, sensitivity of the assay, time required to complete the assay, and cost. Most food and medical rapid assays are limited by the sample preparation time and the assays sensitivity in a “dirty” system (i.e., a system such as a fluid containing solids or slight turbidity). Therefore, there is a need in the art for a rapid assay for microorganisms for use in a relatively “dirty” system. The rapid assay should have the features of high sensitivity and low preparation and assay time.
Many viruses or bacteriophages have been studied, characterized, and used to develop commercial antiviral products. Fluorescent reporters or markers have been bound to bacteriophages as an assay for host bacterial organisms. However, the saturation of the dye or marker on the surface of the bacteriophage and the number of bacteriophage that bind with the host cell is variable. Accordingly, the use of bacteriophages to deliver a marker to a host cell for rapid assay purposes has not been extensively commercialized.
It is known to use peptides derived from bacteriophages in a variety of useful applications. For example, U.S. Pat. No. 6,297,042 for CHEESE MAKING WITH BACTERIOPHAGE RESISTANT BACTERIA, incorporated herein by reference, describes a method for reducing bacteriophage attack on bacteria used in the cheese making industry. The method of the '042 patent comprises addition of “blocker” peptides derived by enzymatic treatment of immunoglobulins, bacteriophages, bacteriophage parts, or mixtures thereof. Similarly, peptides derived by hydrolysis of whey proteins have been used to inhibit culture agglutination (clumping and chaining of cells) (Ustunol, Z. and C. L. Hicks. 1994. Use of an enzyme-treated, whey-based medium to reduce culture agglutination. J. Dairy Sci. 77:1479-1486; Hicks, C. L., C. E. Onuorah, and I. Surjawan. 2000. Use of hydrolyzed whey peptides to inhibit culture agglutination. J. Dairy Sci. 83:1196-1202; both incorporated herein by reference), prevent binding of intact immunoglobulins (Hicks, C. L. and Z. Tabeidie. 1998. Effect of homogenization on immunoglobulins' agglutination response in presence of lactic cultures. J. Dairy Sci. 81(Suppl. 1):32; Tabeidie, Z. and C. L. Hicks. 1998. Effect of heat treatment on immunoglobulins' agglutination response in presence of lactic cultures. J. Dairy Sci. 81(Suppl. 1):32; both incorporated herein by reference), and block bacteriophage adsorption and inhibit bacteriophage proliferation (Hicks, C. L., C. E. Onuorah, and I Surjawan. 2002. Use of hydrolyzed whey peptides to inhibit Lactococcus lactis ssp. cremoris wwa phage proliferation. J. Dairy Sci. 84:Submitted; U.S. Pat. No. 6,297,042; both incorporated herein by reference). Peptides derived from ficin hydrolysis of bacteriophage (φc2) were shown to inhibit growth of Lactococcus lactis ssp. lactis C2 (Hicks, C. L., P. A. Clark-Safko, I. Surjawan, and J. O′Leary. 2004. Use of bacteriophage-derived peptides to delay phage infections. Food Res. Intl. 37 (2):115-122; incorporated herein by reference).
The present invention addresses the identified need in the art by providing bioactive probes comprising peptides labeled with a marker. Typically, the peptides are attachment peptides derived from bacteriophages, or are synthesized analogs or derivatives which correspond to such attachment peptides and to bind to the same receptor molecules. It is known that attachment peptides of various bacteriophages can range across genus (i.e. peptide sequences that bind to calcium receptors), or can be limited to individual strains (i.e. peptide sequences specific to multiple carbohydrate moieties of a particular bacterial cell wall). Thus, peptide-based probes are provided, attached to a marker, which deliver the marker to the surface of a specifically targeted bacteria where the concentration of the marker can be quantified. Further, a method for detecting a bacteria is provided, comprising attaching a detectable molecule to a smaller bacteriophage peptide or synthetic bacteriophage peptide which binds to specific host bacteria, thereby increasing saturation of the marker and improving sensitivity of the assay.